The results show that PCX can inhibit cancer cell migration due to its CXCR4 antagonism. 0.05 as the minimal level of significance. 3. RESULTS AND DISCUSSION 3.1. CXCR4 Expression BAPTA and CXCR4-Mediated Migration in HuCCT1 Cells Surface expression of CXCR4 in HuCCT1 cells was confirmed by circulation cytometry (Physique 1A). Over 36% of the HuCCT1 cells were CXCR4-positive with enhanced fluorescence intensity per cell. We then assessed the involvement of CXCR4 in the migration of the cells. A migration assay was performed to test whether SDF-1 induced migration of HuCCT1 cells and whether this migration could be inhibited by CXCR4 antagonists. As shown in Physique 1, panel B, substantially increased migration across the transwell place membrane was observed in HuCCT1 cells stimulated with the chemoattractant SDF-1. In agreement with previous reports in other cholangiocarcinoma cells, the migration could be significantly inhibited by CXCR4 antagonist AMD3100.30 Open in a separate BAPTA window Determine 1 Characterization of the CXCR4 status of HuCCT1 cells. (A) Circulation cytometric histograms show CXCR4 expression on HuCCT1 cell surface. The percent of CXCR4-positive cells and mean fluorescence intensity were analyzed using FlowJo software. (B) Inhibition of CXCR4-mediated cell migration. HuCCT1 cells were treated with AMD3100 (300 nM) and allowed to migrate through transwell membranes upon activation with SDF-1 for 24 h. Three 20 imaging areas were randomly selected for each place, and each group was conducted in triplicate. Data are shown as mean SD (= 3). ***, 0.001. 3.2. Preparation and Physicochemical Characterization of BAPTA PCX/microRNA Polyplexes The ability of PCX to form polyplexes with microRNA was first evaluated by agarose gel electrophoresis. As shown BAPTA in Physique 2, panel A, PCX was able to fully condense microRNA above a PCX/microRNA (w/w) ratio of 2. PCX condensation of the microRNA was observed already at low w/w ratios (0.5C1) as indicated by the smear of the ethidium bromide-stained microRNA and by the strong fluorescence in the starting well of the gel. At higher PCX/microRNA w/w ratios (above 2), condensed microRNA was completely guarded from ethidium bromide binding, and no fluorescence transmission was observed. The ability of the PCX/microRNA polyplexes to release microRNA was then assessed by heparin displacement assay (Physique 2B). For PCX/microRNA polyplexes prepared at w/w 12, heparin was able to dissociate the polyplexes and completely release microRNA above Rabbit polyclonal to GPR143 200 = 3). Hydrodynamic size and zeta-potential of PCX/microRNA polyplexes prepared at numerous w/w ratios were measured by dynamic light scattering. Polyplexes with all the tested w/w ratios exhibited size in a narrow range from 160C180 nm with polydispersity indexes less than 0.2 (Physique 2C). The size distribution of polyplexes showed a log-transformed normal distribution (Physique 2D). As expected, increasing the w/w ratio used in the preparation of the polyplexes resulted in an increase of the zeta potential (Physique 3E). Open in a separate window Physique 3 Cellular uptake and intracellular trafficking of PCX polyplexes. (A) Overlayed histogram of circulation cytometry analysis of cells treated with PCX/FITC-Oligo polyplexes at numerous w/w ratios (200 nM FITC-Oligo). Quantification of cellular uptake is shown by (B) mean fluorescence intensity (MFI) and (C) % cell uptake. Data are shown as mean SD (= 3). (D) Intracellular trafficking of PCX/FITC-Oligo in HuCCT1 cells by CLSM after 4 h of incubation. 3.3. Cellular Uptake and Intracellular Trafficking To study the cellular uptake and intracellular trafficking of the polyplexes, we used a fluorescently labeled FITC-Oligo (200 nM) instead of microRNA in the preparation of the polyplexes. HuCCT1 cells were treated with PCX/FITC-Oligo for 4 h before circulation cytometry analysis. As shown in Physique 3, panel A, PCX polyplexes exhibited significant cellular uptake in HuCCT1 cells as indicated by the enhanced fluorescence intensity when compared with untreated cells or BAPTA cells treated with free FITC-Oligo. Increasing the w/w ratios in preparation of the polyplexes resulted in enhanced cell uptake both in terms of the imply fluorescence intensity per cell (Physique 3B) and the percentage of cells that have taken up the polyplexes.